Team:Marburg/Labbook/CDI
15/05/18
PCR
| PCR | PCR Mix 1 | PCR Mix 2 |
| GXL buffer | 10 uL | 10 uL |
| dNTPs (from GXL kit | 4 uL | 4 uL |
| GXL polymerase | 2uL | 2uL |
| ICD001 (10 uM) | 0.5uL | 1uL |
| ICD007/ICD008 (10 uM) | 0.5uL | 1uL |
| EC93 genomic DNA (15 ng/uL) | 0.5 uL | 1 uL |
| ddH2O | 32.5uL | 29.5uL |
| Program | time |
| Start cycle (30x) | |
| 98 °C | 10 s |
| 60 °C | 15 s |
| 68 °C | 2 min |
| close cycle | |
| 8 °C | store |
Ran of analytical gel. No bands were observed.
15/05/19
PCR
Bands visible! Lanes 1 and 2 right of the ladder should have a 10506 bp band. Lanes 3 and 4 right of the ladder should have a 11414 bp band (gel description should read EC93 instead of EC90).
PCR cleanup (according to Thermo Scientific protocol), elution in 21 ul.
Concentrations according to Nanodrop: EC93 w/ 001+007: 92 ng/ul; EC93 w/ 001+008: 57 ng/ul
15/05/21
PCR
Primers ICD014 and ICD015 arrived! These will be used to amplify the Pick up backbone with overlaps for the full-length Cdi operon. PCR should yield a 3391 bp fragment.
| PCR | PCR Mix 1 |
| GXL buffer | 10 uL |
| dNTPs (from GXL kit | 4 uL |
| GXL polymerase | 2uL |
| ICD0014 (10 uM) | 1 uL |
| ICD0015 (10 uM) | 1uL |
| 1:10 diluted plasmid X | 1 uL |
| ddH2O | 31 uL |
| Program | time |
| Start cycle (30x) | |
| 98 °C | 10 s |
| 60 °C | 15 s |
| 68 °C | 35 s |
| close cycle | |
| 8 °C | store |
Turned out that the template was wrong. Inoculated 3 ml LB+chloramphenicol for overnight culture of correct Curly clone (clone No. 3) for Miniprep tomorrow.
15/05/22
PCR, Gibson Assembly and Transformation
Miniprep of 1.5 ml of Curly clone No. 3 according to Thermo Scientific Protocol.
PCR with primers ICD014 and ICD015 and freshly prepped Curly plasmid to amplify the backbone for the CDI plasmid (pICD001).
Reaction mix and PCR program see 15-05-21.
Analytical gel: Correct size! (3391 bp)
Gibson assembly of Curly plasmid/ICD014+015 PCR fragment and CDI operon (EC93 genomic DNA with ICD001 +008)
G.A. reaction mix:
| Gibson mix | volume |
| Insert | 2.65ul (= 151ng = 0.02 pmol) |
| Backbone | 0.54ul (= 45ng = 0.02 pmol) |
| G.A. master mix | 10 uL |
| ddH2O | 6.81 uL |
| temperature | time |
| 50 °C | 15 min |
| 16 °C | store |
• Thaw NEB turbo cells on ice
• Dilute G.A. mix 1:3 (5ul mix + 10ul ddH2O)
• Add 2ul of dilution to 25ul of NEB turbo cells
• Transfer the cells to electroporation cuvette
• Electroporate and add 975ul SOC medium immediately
• Recover 1h at 37°C
• Spin down briefly and discard supernatant
• Plate on chloramphenicol plate
15/05/26
PCR, Gibson Assembly and Transformation
On 15-05-22 DpnI digestion has been forgotten, so the transformed cells probably carry the Curly plasmid instead of the Gibson assembly product. So we repeated the procedure including DpnI digestion.
PCR of Curly backbone: See 15-05-21
DpnI digestion program:
| temperature | time |
| 37 °C | 30 min |
| 16 °C | store |
Gibson assembly of Curly plasmid/ICD014+015 PCR fragment and CDI operon (EC93 genomic DNA with ICD001 +008) G.A. reaction mix:
| Gibson mix | volume |
| Insert | 2.65ul (= 151ng = 0.02 pmol) |
| Backbone | 0.51ul (= 45ng = 0.02 pmol) |
| G.A. master mix | 10 uL |
| ddH2O | 6.84 uL |
| temperature | time |
| 50 °C | 15 min |
| 16 °C | store |
• Thaw NEB turbo cells on ice
• Add 2ul of Gibson assembly product to25ul of NEB turbo cells
• Transfer the cells to electroporation cuvette
• Electroporate and add 975ul SOC medium immediately
• Recover 1h at 37°C
• Spin down briefly and discard supernatant
• Plate on chloramphenicol plate (300µL of 0,4% Glucose added to the plate to reduce transcription of Lac promoter)
Gibson mix was not diluted 1:3 in contrast to first attempt on 15-05-22
15/05/27
Plasmidisolation
Colonies are grown wich hopefully carry CDI plasmid. To check this, 12 colonies have been picked and cultivated in 3mL LB broth with additional 0.4% glucose.
In the evening, 1.5ml of cell suspension were pelleted and Miniprep was carried out with the Qiagen Robot.
15/05/28
Plasmidisolation, SacI Digest
The concentration of all the 12 colonies was measured and noted as follows:
| plasmid | concentration ng/uL |
| #1 | 77 |
| #2 | 30 |
| #3 | 90 |
| #4 | 88 |
| #5 | 88 |
| #6 | 86 |
| #7 | 97 |
| #8 | 74 |
| #9 | 84 |
| #10 | 88 |
| #11 | 62 |
| #12 | 81 |
| Digest | volume |
| DNA template | 10 uL |
| Cutsmart buffer | 2 uL |
| Distilled water | 7 uL |
| SacI | 1 uL |
| total | 20 uL |
Analytic gel ; correct size abt 6700 and 8000
The DNA sample was send to determine the sequence and it was positive. We actually got the sequence which we expected.
15/05/31
Overnightcultures
Inoculation of
• four BBa_I13522 (GFP) clones in 3ml LB+CAM overnight
• Olga's W3110 strain with integrated RFP and Kann resistance in 3ml LB+Kann
• W3110 wildtype in 3 ml LB as host for our GFP construct
15/06/1
Transformation into chem. comp. cells
Plasmid purification (four BBa_I13522 (GFP)) was realized using the "Thermo Scientific GeneJET Plasmid Miniprep Kit"
ansformation:
GFP_1 Plasmid into E.coli (strain W3110)
pICD001-1 Plasmid into E.coli (strain W3110 with integrated RFP)
pICD001-3 Plasmid into E.coli (strain W3110 with integrated RFP)
Transformation protocol:
1. Dilute 1:100 overnight culture (Max 15-05-31)
2. Grow 10 mL day culture (2.5 h) at 37 °C
3. Take 1 mL, spin down full speed (1 min, 17.000 rpm), put on ice
4. Add to pellet 100 micro liter of TSS, resuspend on ice.
5. Add 1 micro liter of Plasmid (GFP_1 ; CDI_1; CDI_3)
6. Incubate 45 Min on ice
7. Put at 42 °C for 1 min.
8. Incubate 15 min on ice
9. Add 1 mL LB Medium with 0.4 % Glucose, resuspend, incubate 2 h incubator at 37 °C, 220 rpm
10. Spin down, resuspend in 50 micro liter residual media, plate on LB-Kann, incubate at 37 °C over night (step 10 done by Max)
15/06/2
Transformation into chem. comp. cells
Colonies on all three transformation plates!
• GFP plasmid (BBa_I13522): Colonies beyond count
• pICD001-1 Plasmid into E.coli (strain W3110 with integrated RFP): 23 colonies
• pICD001-3 Plasmid into E.coli (strain W3110 with integrated RFP): 26 colonies
Finally the sequencing of pICD001-1 and pICD001-3 is done! Correct Gibson assembly sequences were checked by sequenceing with standard primers iGEM130-fwd and iGEM160-rev and 100ng/ul pICD001 plasmids. Both plasmids are correct, so pICD001-1 will become pICD001 and pICD001-3 can be trashed.
As a result, the W3110/RFP strain transformed with pICD001-1 will become strain CDI002 (a.k.a. the killer strain). The W3110 wildtype transformed with the GFP plasmid (BBa_I13522) will become strain CDI003 (a.k.a. the Opfer strain).
Inoculation of CDI002 and CDI003 in 3 ml LB CAM +1%glucose
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